Deciphering the organization of noncoding sequences involved in the regulation of eukaryotic gene expression, both structurally and functionally, is a major, post-genome sequence challenge. We believe that developing an understanding of evolutionary processes acting on these sequences, and establishing evolutionary rules governing the relationship between structure/function and evolutionary constraint, will be critical to meeting this challenge. Our proposal is to experimentally dissect evolved changes in a model cis-regulatory element, the Drosophila even-skipped stripe 2 element, one of the best functionally characterized of all eukaryotic enhancer sequences. Our experiments will allow us to determine the functional importance of evolved changes in the sequences of regulatory protein binding sites within the element, the functional importance of changes in spacing between binding sites, and the possible co-evolutionary interaction of the two. In addition, we describe the technical capability to conduct these evolutionary genetic analyses of interspecific differences in the genetic background of each species under investigation, providing the first opportunity to critically examine the importance of trans-acting factor coevolution in the evolution of gene regulation. These experiments will benefit the analysis of eukaryotic genome structure/function and will aid work to decipher the controls of human gene expression.